Directly injectable formulations which provide enhanced cryoprotection of cell products

ABSTRACT

This invention provides compositions and methods for cryoprotection of recombinant live cancer cells. Specifically, an improved cryoprotective medium is provided which includes a hydroxyethyl starch and/or derivative thereof alone or in combination with either DMSO or glycerol.

FIELD OF THE INVENTION

The invention relates to compositions and methods for thecryopreservation of cellular vaccines. More specifically, the inventionrelates to the use of particular compositions comprising cryoprotectivemedia that includes hydroxyethyl starch (HES) and glycerol or DMSOtogether with recombinant live cancer cells. The compositions can befrozen and thawed, when desired, for use as a cellular vaccine for thetreatment of cancer.

BACKGROUND OF THE INVENTION

At certain periods of time in a tumor's cell growth, the patient'simmune system has the ability to recognize the growth as abnormal (ornon-self. As a result, various methods have been developed which takeadvantage of the patient's own immune system to fight cancer. Exemplarymethods include the use of polyclonal and monoclonal antibodies,non-specific immune system stimulants, such as cytokines, protein orpeptide subunit vaccines (e.g., using antigens that are often associatedwith cancer cells, such as tumor-specific antigens and tumor-associatedantigens), adoptive immunotherapy or cellular therapy, gene therapy,cellular vaccines, etc. See, e.g., WO 00/3387.

Cellular vaccines wherein cells (or derivatives thereof are thetherapeutic agent are currently in clinical testing for treatment ofcancer. Such cellular vaccines provide advantages over isolated proteinvaccines in that whole cells are the vehicle for a broad range ofantigens, e.g., on the cell surface. See, for example, Dranoff et al. WO93/06867, Gansbacher et al., WO 94/18995, Jaffee et al WO 97/24132,Mitchell et al. WO 90/03183 and Morton et al WO91/06866, each of whichis expressly incorporated by reference herein.

Cells for use in such cellular vaccines can be modified, e.g. to expressa protein which modulates the immune response to the cell. For example,a gene encoding a cytokine or costimulatory molecule may be introducedinto cells derived from, e.g., a primary tumor taken from a patient, tocreate recombinant cancer cells. When the cytokine or costimulatorymolecule is expressed, it is capable of modulating the immune responseto the cell. The recombinant cancer cells may be expanded in vitro,treated to prevent further growth and returned to the patient.Appropriate timing of administration(s) and good cell viability arerequired for effective treatment with a cellular vaccine. Thus, it isimportant to be able to store recombinant cancer cells for use atselected time points, appropriate to particular treatments. Storage andmaintenance of viability are important in order to allow fortransportation, to decrease the amount of cell divisions the cellsundergo before use in treatment and to ensure that an adequate andreproducible dose is delivered to the patient.

Freezing of cell compositions with maintenance of viability has been thesubject of considerable research. Maintenance of cell viabilityfollowing freezing and thawing continues to be a challenge. In thefreezing process as the liquid component of a cell is changed to asolid, ice crystals are formed and damage can occur to the cells. Atleast two types of damage to cells is possible when ice crystals areformed. Rapid growth of ice crystals may physically disrupt membranesand subcellular organelles and may even lyse cells. Slow growth of icecrystals may result in cellular dehydration (because of the exclusion ofelectrolytes from the ice crystals) and extra-cellular ice formation.See, e.g., Gorlin, (1996) Journal of Infusional Chemotherapy,6(1):23-27.

In an attempt to minimize the effects of ice crystal formation, cellsare typically frozen in medium with cryoprotectants. Cryoprotectantsprotect the cells during freezing in a variety of ways. Collagiativecryoprotectants penetrate the cell and decrease the osmotic gradientacross membranes. Vitrifying cryoprotectants increase the glassformation of the solution thereby creating a glass wall around the cell,which prevents dehydration. Cryoprotectants can also work by inhibitingice crystal formation. See, e.g., Gorlin, (1996) Journal of InfusionalChemotherapy, 6(1):23-27.

Different cell types vary in their permeability to water and in theirsensitivity to solute concentration. Leibo et al., (1970) Cryobiology,6(4): 315-332. As a result, different types and combinations ofcryoprotectants have been found to be effective to preserve specifictypes of cells. For example, human bone marrow committed stem cells havebeen shown to be preserve by a cryoprotectant combination of dextran,glycerol, and dimethyl sulfoxide (Odavic et al. (1980) Experienta36:1122-1124), and mouse marrow stem cells have been shown to bepreserved by polyvinylpyrolidone (PVP), sucrose or glycerol. See, e.g.,Stiff et al. (1987) Blood, 70(4): 974-978; Venkataraman, (1997)Cryobiology, 34:276-283; Wang et al., (1998) Cryobiology, 37:22-29;Merten et al., (1995) Biologicals, 23:185-189; and, Yoshida andTakeuchi, (1991) Cytotechnology 5:99-106. Polymers, such as hydroxyethylstarch (HES), have been used to cryoprotect human monocytes andunfractionated cells for use in bone marrow transplantation. See, e.g.,Takashi et al., (1988) Biophysical Journal, 54:509-518; and Stiff et al.(1987) Blood, 70(4): 974-978.

Cryoprotective media for cellular vaccines has not been reported. Thus,there remains a need for cryoprotective media and procedures that can beused to successfully preserve cellular vaccines for use as therapeuticagents. In view of the above, materials and methods that would providefor successful preservation and recovery of cells with a high percentageviability following freezing and thawing would be highly desirable foruse in cellular vaccines. The present invention addresses this need, aswill be apparent from the detailed description provided herein.

SUMMARY OF THE INVENTION

The invention provides compositions and methods that includecryoprotective media and recombinant live cancer cells for protectingand preserving the cells during freezing and thawing. The cryoprotectivemedium includes a hydroxyethyl starch (HES) or derivative thereof aloneor combination with DMSO or glycerol.

Compositions of the invention include a recombinant live cancer cell ina cryoprotective medium comprising about 5% by weight to about 22% byweight of a hydroxyethyl starch (HES) or derivative thereof alone or incombination with about 1% by weight to about 15% by weight of DMSO orglycerol, and, optionally, from about 0% by weight to about 10% byweight human serum albumin (HSA).

A composition can include about 8% by weight of HES or a derivativethereof and about 5% by weight of DMSO or glycerol, and, optionally,about 2% by weight human serum albumin (HSA).

The HES of the invention can include a variety of molecules. Forexample, HES molecules can be: (a) etherified with hydroxyethyl groupswherein the degree of molecular substitution is from about 0.60-0.80;(b) etherified with hydroxyethyl groups, wherein the degree of molecularsubstitution is from about 0.40-0.60; or a combination of (a) and (b).Other examples of HES, include, but are not limited to HES moleculesthat are: (a) etherified with hydroxyethyl groups wherein the degree ofmolecular substitution is about 0.70; (b) etherified with hydroxyethylgroups, wherein the degree of molecular substitution is from about 0.4to about 0.5; or a combination of (a) and (b). In one embodiment, HESincludes a molecule with (a) a molecular weight of from about 420 toabout 480 kDa; (b) a molecular weight of from about 200 to about 290kDa; or a combination of (a) and (b).

Many types of recombinant live cancer cells, such as patient-derivedtumor cells, autologous cells, allogeneic cells and bystander cells,respectively, can be used in practicing the invention. Typically, arecombinant live cancer cell for use in practicing the inventioncomprises an introduced heterologous nucleic acid coding sequence orgene for a cytokine or costimulatory molecule.

Methods for preserving viability of recombinant live cancer cells arealso included within the invention. Such methods include obtaining aplurality of recombinant live cancer cells, concentrating the cells,suspending the cells in a cryoprotective medium such as described above,thereby providing a suspension and freezing the suspension to acryogenic frozen state, thereby providing a frozen suspension. Afterthawing of the suspension to a liquid state at least about 60%, about65%, about 70%, about 75% or more of the recombinant live cancer cellsremain viable. In one embodiment, the recombinant live cancer cells areirradiated prior to freezing or in the cryogenic frozen state, whereinthe suspension is frozen at a temperature from about −200° C. to atemperature of about −35° C. The methods further include thawing thefrozen suspension and administering the thawed suspension to a subject.Optionally, the thawed suspension is not washed prior to theadministering step.

The invention further includes a frozen or thawed suspension prepared bya method described herein.

Kits for preserving cell viability are also included in the invention.Kits of the invention include: a container containing a cryoprotectivemedium such as described above and instructional materials teaching theuse of the cryoprotective medium to preserve cell viability.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the % viability of LNCaP cells before(pre-freeze), immediately after (initial thaw) and 24 hours after thecells were suspended in ctyoprotective media, frozen to −80° C. andthawed at 37° C. The cryoprotective media tested was as follows: 1× PBSalone; 5% glycerol alone; 24% HSA, 5% glycerol; 5% HSA, 5% glycerol;saturated HES in 5% glycerol/5% HSA/PBS; 20% Hetastarch in 5%glycerol/5% HSA/PBS; 20% Pentastarch in 5% glycerol/5% HSA/PBS; 15% PEG(10K) in 5% glycerol/5% HSA/PBS; Dextran 50K in 5% glycerol/5% HSA/PBS.

FIGS. 2A and 2B are graphs showing the viable cell recovery (%viability) of LNCaP cells either immediately following freezing at −80°C. and thawing at 37° C. (initial thaw) or 24 h after thawing (24 hrlater). The LNCaP cells were suspended in cryoprotective mediacomprising polymer 1B (20% HES) alone with or without a second polymer(FIG. 2A) or polymer 1B (20% HES) plus 5% glycerol with or without asecond polymer (FIG. 2B).

FIGS. 3A and 3B are graphs showing the % viability (FIG. 3A) and GM-CSFsecretion (FIG. 3B), of several independently produced cultures of PC-3cells, each of which was suspended in 20% HES+5% DMSO or 5% glycerol,frozen to −80° C., then thawed at 37° C.

FIGS. 4A and 4B are graphs showing the % viability (FIG. 4A) and GM-CSFsecretion (FIG. 4B) of several independently produced cultures of LNCaPcells, each of which was suspended in 20% HES+5% DMSO or glycerol,frozen at −80° C., then thawed at 37° C. FIG. 5 is a graph showing the %viable cell recovery of LNCaP cells immediately following freezing at−80° C. and thawing at 37° C. or 24 h after thawing. The cells werefrozen in cryoprotective medium comprising from 8% to 26% HES and 5%glycerol in PBS. FIG. 6A is a graph showing the % viability of PC-3cells in a variety of cryoprotective media at time-zero (the time offreezing) and after 3 months, 6 months and 12 months storage at −80° C.The formulations tested are indicated in the figure.

FIG. 6B is a graph showing the GM-CSF secretion by PC-3 cells attime-zero (the time of freezing) and after 3 months, 6 months and 12months storage at −80° C. in a variety of cryoprotective media. Theformulations tested are indicated in the figure.

DETAILED DESCRIPTION

There are many benefits to freezing cells in multiple aliquots andpreserving their viability when thawed. For example, the frozen cellscan be stored and/or transported for future use, e.g., for treatment,such as a cellular vaccine. However, freezing cells and preserving theirviability is not a simple matter. Cell types differ in theirpermeability to water and in their sensitivity to solute concentration,thus, different types and combinations of cryoprotectants are needed topreserve specific types of cells. The invention provides composition andmethods for preserving the viability of recombinant cancer cells using acryoprotective medium that includes a hydroxyethyl starch (HES) or aderivative thereof alone or in combination with DMSO or glycerol.

DEFINITIONS

Unless defined otherwise, all scientific and technical terms areunderstood to have the same meaning as commonly used in the art to whichthey pertain. It is to be understood that this invention is not limitedto the particular methodology, protocols, and reagents described, asthese may vary. For the purpose of the present invention, the followingterms are defined below.

The term “autologous” refers to cancer cells derived from an individualor primary descendents of those cells, wherein the cells may be used asa cellular vaccine for that same individual.

The term “allogeneic” as used herein refers to cancer cells of the sametype being harbored by an individual, but established from a cancer cellline derived from an unrelated individual. Alternatively, an allogeneiccancer cell line is derived from an unrelated tumor type to the tumorharbored by an individual, but which shares common tumor associatedantigens with the tumor of that individual. In general, allogeneicrefers to genetic differences within a species, that is, differencesbetween individuals of the same species. It follows that allogeneiccells for use as a cellular vaccine are genetically dissimilar to thoseof the individual to which they are administered.

The term “bystander” as used herein refers to a mammalian cell line,preferably a human cell line, which naturally lacks majorhistocompatibility class I (MHC-I) antigens and major histocompatibilityclass II (MHC-II) antigens or is modified so that it lacks MHC-Iantigens and MHC-II antigens. Theoretically, any mammalian, preferablyhuman, cell line that is capable of paracrine production of a cytokineor costimulatory molecule can be used. An exemplary bystander cell lineis K562 (ATCC CCL-243; Lozzio et al., (1975) Blood 45(3): 321-334; Kleinet al. (1976) Int. J. Cancer 18: 421-431). Heterologous nucleic acids orgenes that encode a cytokine or costimulatory molecule may be introducedinto bystander cells, and they may be mixed with cancer cells for use ina-cellular vaccine.

By “introduced” is meant the provision to an autologous, allogeneic orbystander cell line of a nucleic acid molecule, e.g., a vector, thatcomprises a heterologous nucleic acid coding sequence or gene for acytokine or costimulatory molecule that either is not expressed in thecell line or, as a result of the provision of the nucleic acid molecule,is now expressed at a greater level. A “vector” encompasses a DNAmolecule, such as a plasmid, virus or other vehicle, which contains oneor more heterologous or recombinant DNA sequences, e.g., a cytokine orcostimulatory molecule gene or coding sequence of interest, under theoperative control of a functional promoter and in some cases an enhanceras well. By recombinant or heterologous with reference to a vector orother DNA sequences merely acknowledges the linkage of DNA sequenceswhich are not typically conjoined as found in nature.

The term “patient-derived tumor cells” refers to cells that have beenrecovered from a tumor taken from a patient. Typically a cell suspensionis created from the isolated tumor. When used as a cellular vaccine,patient-derived tumor cells are treated in a manner effective to stopfurther replication, i.e. the cells are irradiated.

The term “recombinant cancer cell” refers to a cancer cell in which aheterologous nucleic acid coding sequence or gene for a cytokine orcostimulatory molecule have been introduced.

The term “vaccine”, as used herein refers to a cellular composition(e.g., a recombinant live cancer cell) for administration to a patientas part of a therapeutic regimen for the treatment of cancer. Thevaccine typically contains cancer cells, some or all of which have beengenetically modified to express a cytokine or other costimulatorymolecule.

The term “cryogenic frozen state” refers to a temperature at which thecells can be frozen and/or stored for a desired length of time. Forexample a cryogenic frozen state can refer to a temperature, e.g., fromabout −200° C. to a temperature of about −35° C., from about −180° C. toa temperature of about −35° C., from about −150° C. to a temperature ofabout −35° C., from about −200° C. to a temperature of about −50° C.,from about −200° C. to a temperature of about −60° C. and the like.

The term “cryoprotective medium” refers to the medium in which cells aresuspended when frozen and/or thawed. For example, the cryoprotectivemedium described herein includes a hydroxyethyl starch (HES) orderivative thereof alone or in combination with DMSO or glycerol. Thecryoprotective medium can also include human serum albumin (HSA). Theterm “cryoprotective medium” may be used interchangeably with the terms“cryo-protecting formulation” and “cryoprotective formulation”. Thecomponents of the “cryoprotective medium” may be described herein as“polymers”.

The terms “nucleic acid” or “oligonucleotide” or grammatical equivalentsherein refer to at least two nucleotides or analogues thereof,covalently linked together. A nucleic acid of the invention is typicallysingle-stranded or double stranded and will generally containphosphodiester bonds, although in some cases, nucleic acid analogs areincluded that can have alternate backbones, comprising, for example,phosphoramide; phosphorothioate, O-methylphophoroamidite linkages orpeptide nucleic acid backbones and linkages. The depiction of a singlestrand also defines the sequence of the other strand and thus alsoincludes the complement of the sequence.

The term “hydroxyethyl starch” or “HES”, as used herein refers to apolymer of hydroxyethyl starch or a derivative thereof, which exhibitsthe same properties as a cryoprotectant as HES itself. HES of theinvention can include a variety of molecules, as further describedbelow.

The term “patient” as used herein may refer to any mammal. The inventionis useful for both the human and other mammalian subjects.

Cryoprotective Medium

The invention provides cryoprotective formulations for preserving theviability of recombinant live cancer cells during freezing to acryogenic frozen state and thawing to a liquid state. These preservedrecombinant live cancer cells can be used, when desired, as cellularvaccines, e.g., for the treatment of cancer.

Cryoprotective medium of the invention includes at least onecryoprotectant, such as, hydroxyethyl starch (HES), and often includesother cryoprotectants as well, e.g., DMSO, or glycerol, and optionallyhuman serum albumin (HSA). In one embodiment, the cryoprotective mediumof the invention can be free or substantially free of human serumalbumin (HSA). Since HSA is a human-derived material, elimination ofthis excipient can reduce the potential risk of contamination ofcellular product with adventitious agents.

The cryoprotective medium of the invention includes a polymer,hydroxyethyl starch (HES). HES has typically found use as a plasmaexpander. Hetastarch, the most common hydroxyethyl starch, is derivedfrom corn starch and has a molecular weight of about 450,000 daltons.Pentastarch is an analog of Hetastarch with a molecular weight of about264,000 daltons. The HES of the invention includes a variety ofmolecules. For example, HES can comprise HES molecules that are (a)etherified with hydroxyethyl groups where the degree of molecularsubstitution is from about 0.60-0.80; (b) etherified with hydroxyethylgroups, wherein the degree of molecular substitution is from about0.40-0.60; or a combination of (a) and (b). Other examples of HES,include, but are not limited to, HES that is, e.g., (a) etherified withhydroxyethyl groups where the degree of molecular substitution is about0.70; (b) etherified with hydroxyethyl groups, where the degree ofmolecular substitution is from about 0.4 to about 0.5; or a combinationof (a) and (b). In one embodiment, HES includes a molecule with (a) amolecular weight of from about 420 to about 480 kDa; (b) a molecularweight of from about 200 to about 290 kDa; or a combination of (a) and(b). For example, HES can include Hetastarch (B. Braun-McGaw) and/orPentastarch (B. Braun-McGaw). HES has been used as a protective agentfor the cryopreservation of erythrocytes and platelets and when combinedwith DMSO. See, e.g., Ashwood-Smith et al. (1972) Cryobiology 9:441-449.

Dimethylsulfoxide (DMSO) or glycerol are also optionally used in thecryoprotective media of the invention. While the mechanism is not partof the invention, DMSO and glycerol can act by decreasing the osmoticgradient across membranes. These agents maintain an increased volume ofcellular solution and exert colligative (depending on the number ofparticles (and not on the nature of the particles) action within thecells. This avoids an excessive concentration of toxic electrolytes inthe non-frozen cellular solution. If there is enough protective agentcompound, the salt concentration does not rise to a critically damaginglevel until the temperature becomes so low that the damaging reactionsare slow enough to be tolerated by the cells.

In one aspect, a composition of the invention includes a recombinantlive cancer cell in a cryoprotective medium, wherein the cryoprotectivemedium comprises a hydroxyethyl starch (HES) or derivative thereof aloneor in combination with DMSO. In one embodiment, the composition includesabout 5% by weight to about 22% by weight of HES or a derivative thereofand about 1% by weight to about 15% by weight of DMSO, and, optionally,from about 0% by weight to about 10% by weight human serum albumin(HSA). In another embodiment, a composition can include about 5% byweight to about 15% by weight of HES or a derivative thereof and about1% by weight to about 10% by weight of DMSO, and, optionally, from about0% by weight to about 10% by weight human serum albumin (HSA). Inanother embodiment, a composition can include about 5% by weight toabout 10% by weight of HES or a derivative thereof and about 1% byweight to about 6% by weight of DMSO, and, optionally, from about 0% byweight to about 10% by weight human serum albumin (HSA). For example, inone preferred embodiment, a composition of the invention includes arecombinant live cancer cell in a cryoprotective medium comprising afinal percentage of about 8% by weight HES, about 2% by weight HSA, andabout 5% by weight DMSO. HES may include Pentastarch, Hetastarch, acombination or derivative thereof.

Compositions of the invention also include a recombinant live cancercell in a cryoprotective medium, wherein the cryoprotective mediumcomprises a hydroxyethyl starch (HES) or derivative thereof alone or incombination with glycerol. In one embodiment, a composition includesabout 5% by weight to about 22% by weight of the HES or derivativethereof and about 1% by weight to about 15% by weight of the glycerol,and, optionally, from about 0 to about 10% human serum albumin (HSA). Inanother embodiment, a composition includes about 5% by weight to about15% by weight of the HES or derivative thereof and about 1% by weight toabout 10% by weight of the glycerol, and, optionally, from about 0 toabout 10% human serum albumin (HSA). In yet another embodiment, acomposition includes about 5% by weight to about 10% by weight of theHES or derivative thereof and about 1% by weight to about 6% by weightof the glycerol, and, optionally, from about 0 to about 10% human serumalbumin (HSA). For example, in another preferred embodiment, acomposition of the invention includes a recombinant live cancer cell ina cryoprotective medium comprising a final percentage of about 8% byweight HES, about 2% by weight HSA, and about 5% by weight glycerol. HESmay include Pentastarch, Hetastarch, a combination or derivativethereof.

A cryoprotective medium of the invention is typically diluted in asolution, which is at a physiological pH. Exemplary solutions for use inpracticing the invention include, but are not limited to phosphatebuffered saline (PBS), Dulbecco's Modified Eagle's Medium (DMEM), IDMEM,MEM, RPMI 1640, Ham's F-12, Normosol R, lactated Ringer's, Hank'sbalanced salt solution (HBSS), and combinations thereof. In addition,the cryoprotecive medium can contain auxiliary substances, such as,water, saline, pH buffering agents, carriers or excipients, otherstabilizers and/or buffers or other reagents that enhance the viabilityand/or cytokine or other costimulatory molecule expression of therecombinant live cancer cells following the freezing and thawingprocess.

Typically, the cell suspension is frozen at a temperature of from e.g.,from about −200° C. to a temperature of about −35° C., from about −180°C. to a temperature of about −35° C., from about −150° C. to atemperature of about −35° C., from about −200° C. to a temperature ofabout −50° C., from about −200° C. to a temperature of about −60° C. andthe like. In one embodiment, frozen cells are stored at a temperaturebelow which any recrystallization can occur, e.g., the temperature belowthe glass transition of pure water (e.g., less than about −135° C.).Standard methods known in the art are used to freeze the cells, e.g.,containers holding a cryoprotective medium of the invention andrecombinant cells can be immersed in, a solid carbon dioxide and alcoholmixture, or in liquid nitrogen. The containers can also be placeddirectly in a freezer, which is set at a desired temperature, e.g.,about equal to or less than about −35° C. Cryogenic equipment can alsobe used, e.g., a programmed freezer or rate-controlled freezer(Cryo-Med, Mt. Clemens, Mich. or UTL-80, Neslab Instruments Inc.,Portsmouth, N.H.). The frozen cells are typically transferred to afreezer, which is set at a desired temperature, e.g., about equal to orless than about −35° C. Liquid nitrogen (the liquid and/or gas phase)can also be used to freeze and store the cells. Other freezing methodsand apparatus known in the art can also be used in practicing themethods described herein.

Methods for preserving viability of recombinant live cancer cells uponfreezing and thawing are included in the invention. An exemplary methodincludes the steps of obtaining a plurality of recombinant live cancercells (e.g., mammalian cells, such as lung cancer cells, pancreaticcancer cells, prostate cancer cells, kidney cancer cells, myeloma cells,leukemic cells and the like); concentrating the plurality of recombinantlive cancer cells using standard procedures routinely employed in theart; suspending the recombinant live cancer cells in a cryoprotectivemedium comprising a HES or a derivative thereof alone or in combinationwith DMSO and from about 0% by weight to about 10% by weight human serumalbumin (HSA), thereby providing a suspension. The suspension is frozento a cryogenic frozen state, thereby providing a frozen suspension,where following thawing of the suspension to a liquid state at leastabout 60%, 65%, 70%, 75% or more of the recombinant live cancer cellsremain viable. In one approach, the recombinant live cancer cells areirradiated (e.g., irradiated prior to freezing or irradiated in thecryogenic frozen state). Typically, the frozen suspension is frozen at atemperature of from about −200° C. to a temperature of about −35° C.

The recombinant cancer cells are typically expanded in vitro, treated torender the cells proliferation-incompetent and returned to the patient.In one embodiment, the cells are rendered proliferation-incompetent byirradiation. Typically, the cells are plated in a tissue culture plateand irradiated at room temperature using a Cs source and irradiated at adose rate of from about 50 to about 200 rads/min, preferably, from about120 to about 140 rads/min. In a preferred approach, the cells areirradiated with a total dose sufficient to inhibit the majority of cellsfrom proliferating in vitro. Thus, desirably the cells are irradiatedwith a total dose of from about 10,000 to 20,000 rads, optimally, withabout 15,000 rads, such that 100% of the cells are renderedproliferation-incompetent.

In one embodiment, a frozen suspension prepared a method describedherein is included in the invention. In another embodiment, a thawedsuspension prepared by a method described herein is included in theinvention.

In one embodiment, the methods further include thawing the frozensuspension thereby providing a thawed suspension and administering thethawed suspension to a subject. Optionally, the thawed suspension is notwashed prior to the administering step.

Thawing can take place by allowing samples to thaw slowly at, e.g., roomtemperature, or by immersing frozen samples in liquid, e.g., water, at aset temperature, e.g., about 37° C. Cells can also be thawed by mixingthe cells with thawed medium. Optionally, cells can be thawed using aprogrammed freezer.

In one embodiment, the plurality of recombinant live cancer cells areautologous or allogeneic and further comprise an introduced heterologousnucleic acid coding sequence for a cytokine or costimulatory molecule,e.g., granulocyte-macrophage colony stimulating factor (GM-CSF). Inanother embodiment, the recombinant live cancer cells are bystandercells, and the method further comprises thawing the frozen suspension,thereby providing a thawed suspension; providing patient-derived tumorcells, mixing the thawed suspension with patient-derived tumor cells(e.g., irradiated tumor cells), thereby providing a mixed suspension andadministering the mixed suspension to a subject.

Methods of the invention obtaining a plurality of recombinant livecancer cells (e.g., autologous cells, allogeneic cells, bystander cellsand the like); concentrating the plurality of recombinant live cancercells; suspending the plurality of recombinant live cancer cells in acryoprotective medium, comprising about 8% by weight of HES or aderivative thereof, about 2% by weight HSA and about 5% by weight DMSO,thereby providing a suspension; freezing the suspension to a cryogenicfrozen state, thereby providing a frozen suspension; and irradiating theplurality of live recombinant cancer cells or the frozen suspension,where following thawing of the suspension to a liquid state at leastabout 60%, about 65%, about 70%, about 75% or more of the recombinantlive cancer cells remain viable. In one embodiment, the method furtherincludes thawing the frozen suspension, thereby providing a thawedsuspension and administering the thawed suspension to a subject wherethe thawed suspension is not washed prior to the administering step. Athawed suspension prepared by this method is also included in theinvention.

Following freezing and thawing, the thawed recombinant live cancer cellsare returned, delivered, or transferred to the subject. The cells may bedelivered to the tissue site from which they were obtained or to anothersite appropriate to the therapeutic regimen. If desired, the cells canbe grafted onto a tissue, skin, organ, or body system of interest in thesubject using standard and well-known grafting techniques or deliveredto the blood or lymphatic system using standard delivery or transfusiontechniques. Such delivery, administration, or transfer of thawedrecombinant live cancer cells are typically made by using one or more ofthe routes or modes of administration described herein and know in theart. In one aspect, the amount of thawed recombinant live cancer cellsadministered is sufficient and effective to treat the disease orcondition at the site or tissue system. The thawed recombinant livecancer cells can be administered, for example, intramuscularly,intradermally, subdermally, subcutaneously, orally, intraperitoneally,intrathecally, intravenously, or placed within a cavity of the body(including, e.g., during surgery), or by inhalation or vaginal or rectaladministration.

Typically, the thawed recombinant live cancer cells (s) are administeredin an amount sufficient to induce or reduce a desired phenotype, i.e.,an “effective amount”. Single or multiple administrations of the thawedrecombinant live cancer cells can be carried out as needed. The subjectcan be at any stage of development at the time of administration, e.g.,embryonic, fetal, infantile, juvenile or adult.

Recombinant Cancer Cells

The recombinant live cancer cells used in practicing the invention findutility in active immunotherapy which involves the injection of canceror tumor cells in vivo to generate either a novel or an enhanced immuneresponse thereto. The tumor cells employed can be patient-derived tumorcells (autologous or allogeneic), or bystander cells. Many types ofrecombinant live cancer cells find use in practicing the invention.Typically, the recombinant live cancer cells include an introducedheterologous nucleic acid coding sequence or gene for a cytokine orcostimulatory molecule. Exemplary cytokines or costimulatory moleculesinclude, but are not limited to, any molecule which is involved with theinitiation, enhancement, strengthening, heightening, and/or lengtheningof an immune response. In some embodiments, the cytokine orcostimulatory molecule is GM-CSF, IL-1, IL-2, IL-3, IL4, IL-6, IL-7,IL-10, CD2, IL-12, IL-15, IL-18, TGF, B7, MIP-1a, MIP-1p, MIP-2, M-CSF,G-CSF, and/or ICAM. In one preferred embodiment, the cytokine is GM-CSF.In other embodiments, more than one cytokine or costimulatory moleculeis expressed by a recombinant live cancer cell of the invention.

In one embodiment, a heterologous nucleic acid coding sequence or geneis introduced into a cancer cell, e.g., a lung cancer cell, a pancreaticcancer cell, a prostate cancer cell, a kidney cancer cell, a myelomacell, a leukemic cell and the like. Various methods can be employed fordelivering a nucleic acid molecule, e.g., a vector, to a cell in vitroor ex vivo. Live cancer cells of the invention are typically geneticallyengineered (e.g., transformed, transduced or transfected) with aheterologous nucleic acid coding sequence. Many approaches forintroducing nucleic acids into cells are known in the art. Such methodsinclude electroporation, membrane fusion with liposomes (lipofection),high velocity bombardment with DNA, calcium phosphate mediatedtransfection, DEAE-dextran mediated transfection, infection with viralvectors, by use of polycation compounds such as polylysine, receptorspecific ligands direct microinjection into single cells, spheroplastfusion whereby E. coli containing the nucleic acid molecules arestripped of their outer cell walls and fused to animal cells usingpolyethylene glycol and the like. See, Berger and Kimmel, Guide toMolecular Cloning Techniques, Methods in Enzymology volume 152 AcademicPress, Inc., San Diego, Calif. (“Berger”), Sambrook et al., MolecularCloning—A Laboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring HarborLaboratory, Cold Spring Harbor, New York, 2000 (“Sambrook”), and CurrentProtocols in Molecular Biology, F. M. Ausubel et al., eds., John Wiley &Sons, Inc., (supplemented through 2002), each of which is expresslyincorporated by reference herein.

In vectors for use in practicing the invention, the heterologous nucleicacid coding sequence or gene for a cytokine or costimulatory molecule isoperably linked to a promoter that is capable of driving expression ofthe coding sequence or gene. A coding sequence or gene is “operablylinked” when the promoter is capable of directing transcription of thecoding sequence or gene. A “gene” is any nucleic acid sequence codingfor a protein or mRNA molecule. A gene comprises coding sequences andnon-coding (e.g., regulatory) sequences, while a “coding sequence” islimited to coding DNA. A “promoter” is a DNA sequence that directs thebinding of RNA polymerase and thereby promotes RNA synthesis.“Enhancers” are cis-acting elements of DNA that stimulate or inhibittranscription of adjacent genes. All proper transcription, translationand processing signals (e.g., splicing and polyadenylation signals) arecorrectly arranged on the vector such that the cytokine or costimulatorymolecule gene or coding sequence is appropriately transcribed andtranslated in the cell into which it is introduced. The consrution ofsuch vectors for effective expression in host cells is well within theknowledge of the ordinary skilled artisan.

As used herein, a cytokine or costimulatory molecule gene or codingsequence includes genomic or cDNA sequences, variant sequences andmutations thereof, whether isolated from nature or synthesized in wholeor in part, so long as the gene or coding sequence can express a proteinhaving the characteristic function of the cytokine costimulatorymolecule. The means of modifying genes or coding sequences arewell-known in the art.

Any vector can be employed that is suitable for introduction of nucleicacids into eukaryotic cells, or more particularly animal cells, such asmammalian, e.g., human, cells. Preferably, the vector is compatible withthe cell, e.g., is capable of effecting expression of the cytokine orcostimulatory gene or coding sequence in the cell. Exemplary vectors foruse in practicing the invention include, but are not limited to,viruses, plasmids, retrotransposons, cosmids and/or the like. Viralvectors include, but are not limited to parvovirus vectors, herpes virusvectors, retrovirus vectors, adenovirus vectors, lentiviral vectors, andthe like. Alone, or in combination with viral vectors, a number ofnon-viral vectors are also useful for introducing (e.g., transfecting)heterologous nucleic acid coding sequences for a cytokine orcostimulatory molecule into cells. Suitable non-viral vectors include,but are not limited to, plasmids, cosmids, and phagemids, liposomes,water-oil emulsions, polethyleneimines, biolistic pellets/beads, anddendrimers.

In one aspect of the invention, ex vivo methods are employed wherein aplurality of cells are taken from an individual, a heterologous nucleicacid coding sequence or gene for a cytokine or costimulatory molecule isintroduced into the cells in a manner effective to express the cytokineor costimulatory molecule, as further described above.

Ex vivo methods are typically employed with cells that are autologous orallogeneic.

A heterologous nucleic acid coding sequence or gene for a cytokine orcostimulatory molecule may be introduced into such autologous orallogeneic cells, followed by expression thereof, resulting inproduction of a recombinant live cancer cell. The autologous orallogeneic recombinant live cancer cell can be frozen using thecryoprotective medium and methods described herein. When thawed, therecombinant live cancer cells can be administered to a patient as acellular vaccine.

Bystander cells are cells that lack major histocompatibility class I(MHC-I) antigens and major histocompatibility class 11 (MHC-II) antigenson their surface and hence find use as a universal cytokine orcostimulatory molecule-producing cell line. A heterologous nucleic acidcoding sequence or gene for a cytokine or costimulatory molecule may beintroduced into such bystander cells, followed by expression thereof,resulting in production of a recombinant live bystander cell line. Thebystander cell line can be frozen with the cryoprotective medium andmethods described herein. When thawed, the recombinant bystander cellscan be administered to a patient as a cellular vaccine. The use ofbystander cells in active immunotherapy is described in U.S. Pat. No.6,464,973, expressly incorporated by reference herein.

The recombinant live cancer cells can be cultured in conventionalnutrient media modified as appropriate to the particular cell type.Culture methods and modifications thereof are well within the knowledgeof those of skill in the art. References for cell isolation and cultureinclude Freshney (2000) Culture of Animal Cells, a Manual of BasicTechnique, fourth edition, Wiley-Liss, New York and the references citedtherein; Payne et al. (1993) Plant Cell and Tissue Culture in LiquidSystems John Wiley & Sons, Inc. New York, N.Y.; Gamborg and Phillips(eds.) (1995) Plant Cell, Tissue and Organ Culture; Fundamental MethodsSpringer Lab Manual, Springer-Verlag (Berlin Heidelberg N.Y.) and Atlasand Parks (eds.) The Handbook of Microbiological Media, second edition(1997) CRC Press, Boca Raton, Fla.

Kits

Kits for preserving the viability of recombinant live cancer cells arealso included in the invention. In one aspect, a kit of the inventionincludes: a container containing a cryoprotective medium comprisingabout 5% by weight to about 22% by weight hydroxyethyl starch (HES) orderivative thereof alone or in combination with about 1% by weight toabout 15% by weight DMSO, and, optionally, from about 0 to about 10%human serum albumin (HSA), and instructional materials teaching the useof the cryoprotective medium to preserve cell viability. In anotheraspect, a kit of the invention includes: a container containing acryoprotective medium comprising about 5% by weight to about 15% byweight hydroxyethyl starch (HES) or derivative thereof alone or incombination with about 1% by weight to about 10% by weight DMSO, and,optionally, from about 0 to about 10% human serum albumin (HSA), andinstructional materials teaching the use of the cryoprotective medium topreserve cell viability. In yet another aspect, a kit of the inventionincludes: a container containing a cryoprotective medium comprisingabout 5% by weight to about 10% by weight hydroxyethyl starch (HES) orderivative thereof alone or in combination with about 1% by weight toabout 6% by weight DMSO, and, optionally, from about 0 to about 10%human serum albumin (HSA), and instructional materials teaching the useof the cryoprotective medium to preserve cell viability.

A kit can also include a container containing a cryoprotective mediumcomprising about 5% by weight to about 22% by weight hydroxyethyl starch(HES) or derivative thereof, about 3% by weight to about 15% by weightglycerol, and, optionally, from about 0 to about 10% human serum albumin(HSA), and, instructional materials teaching the use of thecryoprotective medium to preserve cell viability. In another embodiment,a kit can also include a container containing a cryoprotective mediumcomprising about 5% by weight to about 15% by weight hydroxyethyl starch(HES) or derivative thereof, about 3% by weight to about 10% by weightglycerol, and, optionally, from about 0 to about 10% human serum albumin(HSA), and, instructional materials teaching the use of thecryoprotective medium to preserve cell viability. In yet anotherembodiment, a kit can also include a container containing acryoprotective medium comprising about 5% by weight to about 10% byweight hydroxyethyl starch (HES) or derivative thereof, about 3% byweight to about 6% by weight glycerol, and, optionally, from about 0 toabout 10% human serum albumin (HSA), and, instructional materialsteaching the use of the cryoprotective medium to preserve cellviability.

The HES in the kit can include a variety of molecules as describedherein. In one embodiment, HES includes HES, which is, e.g., (a)etherified with hydroxyethyl groups wherein the degree of molecularsubstitution is about 0.70; (b) etherified with hydroxyethyl groups,wherein the degree of molecular substitution is from about 0.4 to about0.6; or a combination of (a) and (b). In another embodiment, HESincludes a molecule with, e.g., (a) a molecular weight of from about 420to about 480 kDa; (b) a molecular weight of from about 200 to about 290kDa; or a combination of (a) and (b).

The instructional material can be affixed to the packaging material orcan be included as a package insert. While the instructional materialtypically comprise written or printed materials they are not limited tosuch. Any medium capable of storing such instructional material andcommunicating them to an end user is contemplated by this invention.Such media include, but are not limited to, electronic storage media(e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g.,CD ROM), and the like. As used herein, the term “instructional material”can include the address of an internet site that provides theinstructions.

The following examples are offered to illustrate, but not to limit theclaimed invention.

EXAMPLE 1

Viability And Cytokine Production Following Freezing And Thawing OfRecombinant Live Cancer Cells

The present invention provides formulations and methods for thecryopreservation of recombinant live cancer cells. The cells may beautologous, allogeneic or bystander cells. The formulations and methodsdescribed herein are applicable to all three types of recombinant livecancer cells. In developing the methods and formulations describedherein, the allogeneic human prostate tumor cell line, LNCaP, wascultured under standard conditosn, pelleted and resuspended in a numberof different cryoprotective media. LNCaP cells were suspended in eachcryoprotective medium, frozen to −80° C., then thawed in a 37° C. waterbath, washed with culture medium. Viability was evaluatedmicroscopically by hemacytometer using the trypan-exclusion method. FIG.1 is a graph showing the % viability of LNCaP cells before (pre-freeze),immediately after (initial thaw) and 24 hours after the cells weresuspended in cryoprotective media, frozen to −80° C. and thawed at 37°C. Viability of greater than about 60% was maintained when thecryoprotective media included HES (either Hetastarch or Pentastarch)together with 5% glycerol and 5% HSA in PBS.

The viability of LNCaP cells following suspension in polymer 1B (20%HES) with and without 5% glycerol and with or without a second polymerwas evaluated microscopically by hemacytometer using thetrypan-exclusion method. The suspensions were frozen to −80° C., thawedin a 37° C. water bath, washed with culture medium, then viability wasevaluated immediately upon thawing at 37° C. and 24 h later. The LNCaPcells were suspended in cryoprotective media comprising polymer 1B (20%HES) alone with or without a second polymer (FIG. 2A) or polymer 1B (20%HES) plus 5% glycerol and with or without a second polymer (FIG. 2B).Viability of greater than about 60% was maintained upon initial thaw and24 hours later in each case where 20% HES was used in combination with5% glycerol in the cryoprotective media. The viability and GM-CSFsecretion of several independently produced cultures of PC-3 prostateadenocarcinoma cells was evaluated following suspension in 20% HES+5%DMSO or glycerol, and freezing at −80° C. The frozen cell suspensionswere thawed in a 37° C. water bath, washed with culture medium, thenviability was evaluated. One million of the medium-suspended cells wereseeded into a culture flask and allowed to attach over 24 hr. At 24 hr,the medium was replaced. At 48 hr, the medium was collected and measuredfor GM-CSF concentration by ELISA. The amount of secreted GM-CSF wasnormalized to a secretion rate per million cells. The % viability of thePC-3 cells is shown in FIG. 3A and GM-CSF secretion by the cells isshown in FIG. 3B. Greater than 80% viability was maintained when eitherDMSO or glycerol was used in the cryoprotective media and a GM-CSFsecretion level of greater than 150 ng/10⁶ cells/day was observed ineach case.

The viability and GM-CSF secretion of several independently producedcultures of LNCaP cells was evaluated following suspension in 20% HES+5%DMSO or glycerol, and freezing at −80° C. The frozen cell suspensionswere thawed in a 37° C. water bath, washed with culture medium, thenviability was evaluated. One million of the medium-suspended cells wereseeded into a culture flask and allowed to attach over 24 hr. At 24 hr,the medium was replaced. At 48 hr, the medium was collected and measuredfor GM-CSF concentration by ELISA. The amount of secreted GM-CSF wasnormalized to a secretion rate per million cells. The % viability of theLNCaP cells is shown in FIG. 4A and GM-CSF secretion by the cells isshown in FIG. 4B. Greater than 60% viability was maintained and a GM-CSFsecretion level of greater than 10 ng/10⁶ cells/day was detected whenDMSO was used in the cryoprotective media.

In a further study, LNCaP cells were suspended in individualcryoprotective media containing 5% glycerol in PBS and from 8% to 26%HES, respectively, then frozen at −80° C. The frozen cell suspensionswere thawed in a 37° C. water bath, washed with culture medium, thenviability was evaluated. The cells were then seeded into a flask withcell culture medium. After 24 hr, the cells were collected and measuredfor viability. FIG. 5 is a graph showing the % viable cell recovery ofthe LNCaP cells immediately following thawing at 37° C. (initial thaw)and 24 h later. The results show that greater than 60% viability wasmaintained when 5% glycerol and from about 10% to 26% HES (Pentastarch)was included in the cryoprotective media.

EXAMPLE 2

Viability And Cytokine Production Following Long Term Freezing AndThawing Of Recombinant Live Cancer Cells

The effect of various cryoprotective media on viability and GM-CSFproduction by recombinant PC-3 cells was evaluated at time-zero (thetime of freezing) and after 3 months, 6 months and 12 months storage at−80° C., respectively. The formulations tested included 24% human serumalbumin (HSA), 5% glycerol; 8% HSA, 10% DMSO, 1% Pentastarch; 8% HSA,10% DMSO; 8% HSA, 10% DMSO, 5% Pentastarch; 8% HSA, 5% DMSO; 20%Pentastarch; 10% DMSO; and 20% Pentastarch, 5% DMSO. At intervals overthe 1 year of storage, the frozen cell suspensions were thawed in a 37°C. water bath, washed with culture medium, then measured for viability.One million of the medium-suspended cells were seeded into a cultureflask and allowed to attach over 24 hr. At 24 hr, the medium wasreplaced. At 48 hr, the medium was collected and measured for GM-CSFconcentration by ELISA. The amount of secreted GM-CSF was normalized toa secretion rate per million cells. The % viability of the cells isshown in FIG. 6A and GM-CSF secretion by the cells is shown in FIG. 6B.Greater than 60% viability was maintained with each cryoprotective mediatested. The most consistent GM-CSF secretion over time was detected when5 or 10% DMSO was used in combination with Pentastarch with or withoutHSA in the cryoprotective media (greater than 200 ng GM-CSF/10⁶cells/day).

All publications, patents, and patent applications cited herein arehereby incorporated by reference in their entirety for all purposes.While this invention has been described with an emphasis upon preferredembodiments, it will be obvious to those of ordinary skill in the artthat variations of the preferred embodiments can be used and that it isintended that the invention be practiced otherwise than as specificallydescribed herein. Accordingly, this invention includes all modificationsencompassed within the spirit and scope of the invention as defined bythe following claims.

1. A composition comprising a recombinant live cancer cell in acryoprotective medium, wherein the cryoprotective medium comprises about5% by weight to about 22% by weight of a hydroxyethyl starch (HESS) orderivative thereof and about 1% by weight to about 15% by weight ofDEMOS.
 2. The composition of claim 1, further comprising from about 0%by weight to about 10% by weight human serum albumin (HAS).
 3. Thecomposition of claim 2, wherein the HESS comprises HESS: (a) etherifiedwith hydroxyethyl groups wherein the degree of molecular substitution isfrom about 0.60-0.80; (b) etherified with hydroxyethyl groups, whereinthe degree of molecular substitution is from about 0.40-0.60; or acombination of (a) and (b).
 4. The composition of claim 3, wherein theHESS comprises HESS: (a) etherified with hydroxyethyl groups wherein thedegree of molecular substitution is about 0.70; (b) etherified withhydroxyethyl groups, wherein the degree of molecular substitution isfrom 0.4 to about 0.5; or a combination of (a) and (b).
 5. Thecomposition of claim 2, wherein the HES comprises HES with: (a) amolecular weight of from about 420 to about 480 kDa; (b) a molecularweight of from about 200 to about 290 kDa; or a combination of (a) and(b).
 6. The composition of claim 2, wherein the recombinant live cancercell comprises an introduced heterologous nucleic acid coding sequencefor a cytokine or costimulatory molecule.
 7. The composition of claim 6,wherein the cytokine is granulocyte-macrophage colony stimulating factor(GM-CSF).
 8. The composition of claim 2, wherein the cancer cell isselected from the group consisting of a lung cancer cell, a pancreaticcancer cell, a prostate cancer, a kidney cancer cell, a myeloma cell,and a leukemic cell.
 9. The composition of claim 2, wherein therecombinant live cancer cell composition comprises patient-derived tumorcells.
 10. The composition of claim 9, wherein said recombinant livecancer cell is autologous.
 11. The composition of claim 2, wherein saidrecombinant live cancer cell is allogeneic.
 12. The composition of claim2, wherein said recombinant live cancer cell is a bystander cell.
 13. Acomposition comprising a recombinant live cancer cell in acryoprotective medium, wherein the cryoprotective medium comprises about8% by weight HES, about 2% by weight HSA, and about 5% by weight DMSO.14. A composition comprising a recombinant live cancer cell in acryoprotective medium, wherein the cryoprotective medium comprises about5% by weight to about 22% by weight of the a hydroxyethyl starch (HES)or derivative thereof and about 1% by weight to about 15% by weight ofthe glycerol, said composition further comprising from about 0% to about10% human serum albumin (HSA).
 15. The composition of claim 14, whereinthe HES comprises HES: (a) etherified with hydroxyethyl groups whereinthe degree of molecular substitution is from about 0.60-0.80; (b)etherified with hydroxyethyl groups, wherein the degree of molecularsubstitution is from about 0.40-0.60; or a combination of (a) and (b).16. The composition of claim 15, wherein the HES comprises HES: (a)etherified with hydroxyethyl groups wherein the degree of molecularsubstitution is about 0.70; (b) etherified with hydroxyethyl groups,wherein the degree of molecular substitution is from 0.4 to about 0.5;or a combination of (a) and (b).
 17. The composition of claim 14,wherein the HES comprises HES with: (a) a molecular weight of from about420 to about 480 kDa; (b) a molecular weight of from about 200 to about290 kDa; or a combination of (a) and (b).
 18. The composition of claim14, wherein the recombinant live cancer cell comprises an introducedheterologous nucleic acid coding sequence for a cytokine orcostimulatory molecule.
 19. The composition of claim 18, wherein thecytokine is granulocyte-macrophage colony stimulating factor (GM-CSF).20. The composition of claim 14, wherein the cancer cell is selectedfrom the group consisting of a lung cancer cell, a pancreatic cancercell, a prostate cancer cell, a kidney cancer cell, a myeloma cell, anda leukemic cell.
 21. The composition of claim 14, wherein saidrecombinant live cancer cell composition comprises patient-derived tumorcells.
 22. The composition of claim 21, wherein said recombinant livecancer cell is autologous.
 23. The composition of claim 14, wherein saidrecombinant live cancer cell is allogeneic.
 24. The composition of claim14, wherein said recombinant live cancer cell is a bystander cell.
 25. Acomposition comprising a recombinant live cancer cell in acryoprotective medium, wherein the cryoprotective medium comprises about8% by weight HES, about 2% by weight HSA, and about 5% by weightglycerol. 26-44. (canceled)
 45. A kit for preserving cell viabilitycomprising: a container containing a cryoprotective medium comprisingabout 5% by weight to about 22% by weight hydroxyethyl starch (HES) orderivative thereof alone or in combination with about 1% by weight toabout 15% by weight DMSO; from about 0% to about 10% human serum albumin(HSA), and instructional materials teaching the use of thecryoprotective medium to preserve cell viability.
 46. The kit of claim45, wherein the HES comprises HES: (a) etherified with hydroxyethylgroups wherein the degree of molecular substitution is about 0.70; (b)etherified with hydroxyethyl groups, wherein the degree of molecularsubstitution is from 0.4 to about 0.5; or a combination of (a) and (b).47. The kit of claim 45, wherein the HES comprises HES with: (a) amolecular weight of from about 420 to about 480 kDa; (b) a molecularweight of from about 200 to about 290 kDa; or a combination of (a) and(b).
 48. A kit for preserving cell viability comprising: a containercontaining a cryoprotective medium comprising about 5% by weight toabout 22% by weight hydroxyethyl starch (HES) or derivative thereof,about 3% by weight to about 15% by weight glycerol; from about 0% toabout 10% human serum albumin (HSA), and, instructional materialsteaching the use of the cryoprotective medium to preserve cellviability.
 49. The kit of claim 48, wherein the HES comprises HES: (a)etherified with hydroxyethyl groups wherein the degree of molecularsubstitution is about 0.70; (b) etherified with hydroxyethyl groups,wherein the degree of molecular substitution is from 0.4 to about 0.5;or a combination of (a) and (b).
 50. The kit of claim 48, wherein theHES comprises HES with: (a) a molecular weight of from about 420 toabout 480 kDa; (b) a molecular weight of from about 200 to about 290kDa; or a combination of (a) and (b).